In most nonexcitable cells, the depletion of internal Ca2+ stores leads to the activation of a plasma membrane Ca2+ entry pathway which plays an important role both for refilling internal Ca2+ stores and for maintaining a prolonged elevation of cytosolic Ca2+ concentration. At present, the mechanism by which depletion of internal stores signals the activation of this Ca2+ entry pathway is not fully understood. However, work from our laboratory suggests that tyrosine kinases play a role in this signalling pathway and points to c-src as a prime candidate for the tyrosine kinase involved. In this proposal, we describe experiments to test the hypothesis that c-src is the tyrosine kinase coupling Ca2+ store depletion to activation of a Ca2+ entry pathway. We will test for involvement of c-src by varying the level of c-src expression and investigating the impact of these changes on Ca2+ entry stimulated by bradykinin and Ca2+ pool depletion. We will use expression antisense techniques and fibroblasts from c-src negative, transgenic mice to test for the effect of reducing c- src levels. We will also express v-src and determine whether this can activate Ca2+ entry in the absence of bradykinin stimulation ofr Ca2+ pool depletion We will continue our investigation of the stimulation of tyrosine kinase activity following Ca2+ pool depletion with thapsigargin. Since our preliminary experiments suggest that c-src may be activated in response to Ca2+ pool depletion, we will focus initially on c-src. We will immunoprecipitate c-src from control cells and cells whose Ca2+ stores have been depleted by a variety of methods and measure c-src activity by in vitro kinase assays. Our hypothesis is that Ca2+ store depletion leads to the activation of c-src tyrosine kinase activity. We describe electrophysiological experiments to characterize the channels activated by bradykinin, Ca2+ pool depletion and overexpression of c-src. We propose to test the hypothesis that a channel with properties similar to the Ca2+-release-activated Ca2+ channel (Icrac), previously described in mast cells and lymphocytes, is activated in fibroblasts by both bradykinin stimulation and Ca2+ store depletion via a mechanism that is dependent on activation of c-src.